Abstract In the third grant cycle of [unreadable]leptin transport across the BBB[unreadable], we will focus on the role of leptin receptor (ObR)- positive astrocytes in relaying leptin from blood to the CNS by crossing the blood-brain barrier (BBB). We hypothesize that astrocytes not only regulate leptin transport as vital components of the BBB, but also modulate neuronal leptin signaling by enabling a more rapid onset and faster termination of leptin action in neurons. The cellular studies will use a co-culture model of primary cerebral microvascular endothelial cells (PBMEC) and astrocytes. We will determine the effects of astrocytes on the permeability and integrity of fluorescently or radioactively labeled leptin and the permeability markers dextran and albumin across the tight monolayer of PBMEC. The mouse studies will test the effects of obesity-induced increase of astrocytic ObR expression, inhibition of astrocyte metabolic activity, and specific deletion of ObR from astrocytes on leptin distribution and neuronal activation in response to leptin. In Aim 1, we will test the hypothesis that astrocytes thwart leptin transport across the PBMEC monolayer by slowing down permeation kinetics. The transcellular rather than paracellular pathways will be shown by morphological examination of the location of fluorescently labeled leptin, as well as the permeation kinetic studies to test the specificity of transport. The effect the level of astrocytic ObR expression will be tested. The transport of leptin will be compared with the distribution of co-administered paracellular permeability markers and immunocytochemistry of tight junction proteins. In Aim 2, we will test the hypothesis that astrocytes and astrocytic ObR participate in the degradation of leptin within the CNS, modulate the half-life of leptin in neurons, and influence neuronal leptin signaling by generation of soluble glial transmitters. Aim 3 will focus on regulatory changes in adult mice with diet-induced obesity or the Avy mutation, both of which have been shown to have regional specific increases of astrocytic ObR. By use of glial metabolic inhibitors and newly generated astrocyte-specific ObR knockout mice, we will show that these ObR(+) astrocytes play an essential role in the regulation of neuronal leptin signaling. The results will provide the first evidence of the functions of ObR(+) astrocytes in linking BBB transport to the CNS response to leptin. An understanding of the consequence of astrogliosis and upregulation of astrocytic ObR in obesity should enable the targeting of astrocytes to counteract the neuroendocrine dysregulation in obese subjects.